MDM2 and XIAP are important cell-survival proteins in tumor cells. MDM2 acts as an oncoprotein, promoting cancer progression mainly through inhibition of the tumor suppressor p53, while the anti-apoptotic protein XIAP plays a critical role in development of resistance to treatment via inhibition of therapy-induced apoptosis. MDM2 overexpression and upregulated XIAP have been detected in various human cancers but not in normal cells/tissues, and elevated MDM2 and XIAP expression in tumor cells is associated with disease progression and poor treatment outcomes. Our previous studies elucidated a molecular mechanism by which the MDM2 C-terminal RING domain interacts with XIAP IRES mRNA resulting in stabilization of MDM2 protein and enhanced translation of XIAP; this led to concomitantly increased expression of both MDM2 and XIAP, contributing to cancer progression and drug resistance. We have recently established a fluorescence polarization (FP) assay for use in high-throughput screening (HTS) of chemical libraries and identified a compound (MX69) that binds to the MDM2 RING domain and blocks or disrupts its interaction with XIAP IRES mRNA. Blocking this interaction results in simultaneous inhibition of both MDM2 and XIAP, leading to cancer cell apoptosis and death. The overall goal of this proposal is to develop a potential novel targeted agent based on the MX69 scaffold against tumors overexpressing MDM2. As discussed above, these tumors also typically upregulate XIAP in an MDM2-dependent manner, resulting in enhanced drug resistance. Specifically, we will perform computer-aided drug design based on the MX69 structure and iteratively optimize MDM2 binding and anticancer activity (Aim 1). We will define the molecular and biological mechanism(s) of action of novel MX69 analogs by solving the X-ray crystal structures of MDM2 in complex with diverse MX69 analogs to confirm on-target MDM2/XIAP inhibition and to evaluate any potential nonspecific effects (Aim 2). We will perform preclinical studies to assess compound stability, pharmacokinetic (PK), and pharmacodynamic (PD) properties of the best MX69 analogs; to evaluate their anticancer activity in vivo using human cancer-in-mouse models; and to determine any potential toxicity to normal cells/tissues (Aim 3). Upon completion of this project, we will have determined the feasibility of dual targeting MDM2/XIAP as a novel therapeutic mechanism, and will have developed promising small molecule inhibitors for further preclinical evaluations, not only for leukemia and neuroblastoma as studied in this proposal, but also in other cancer types.